Corn Peptide Preparation With Coupled Immobilized Enzymolysis And Membrane Separation Assisted By Ultrasound,and In Situ Real-time Monitoring Of Near Infrared Spectroscopy Of Processing

In order to reduce the waste of corn flour as a by-product of corn processing and improve its utilization rate,this study prepared corn ACE inhibitory peptide by using corn protein as the raw material and the preparation method with coupled immobilized enzymolysis and membrane separation assisted by multi-frequency ultrasound.In this way,the concentration and ACE inhibition rate of corn peptides are improved.At the same time,this research studied the effects of multi-frequency ultrasonic treatment and enzymatic hydrolysis on proteins,immobilized enzymes and product peptides at the molecular level.In addition,the research constructed the prediction models of peptide concentration and ACE inhibition rate during ACE inhibitory peptide preparation process using the combination of near infrared spectroscopy and chemometrics.In situ real-time monitoring system was established to provide the basis for intelligent production of functional peptides.The main research results in this work were as follows:(1)Through single factor and response surface methodology experiments,the optimal conditions of corn ACE inhibitory peptides were obtained as follows:frequency combination of 20/28/40 kHz,power density of 150 W/L,duty ratio of 66.7%,enzyme amount of 6320 U/g,pH value of 8.5,temperature of 40^oC,peptide flow rate of 7mL/min,ultrasound time of 40 min,and enzymolysis time of 130 min under the preparation method with coupled immobilized enzymolysis and membrane separation assisted by multi-frequency ultrasound.This method was the best among the four preparation methods,and the protein conversion rate,peptide concentration,ACE inhibition rate and IC₅₀ value were 43.15%,2.01 mg/mL,52.78%and 1.38 mg/mL,respectively.Compared to the preparation method of enzymolysis without multi-frequency ultrasound and off-line membrane separation,the protein conversion rate,peptide concentration and ACE inhibition rate were increased by 13.4%,9.8%and17.8%,respectively,and the IC₅₀ value was decreased by 10.4%.It was concluded that the preparation method with coupled immobilized enzymolysis and membrane separation assisted by multi-frequency ultrasound was more conducive to improving the concentration and ACE inhibitory rate of corn peptides.(2)The results of ultraviolet absorption and fluorescence spectra showed that both enzymatic hydrolysis and multi-frequency ultrasonic treatment changed the conformation of protein molecules.The results of NIR spectroscopy showed that the enzymatic hydrolysis and multi-frequency ultrasonic treatment both weakened the molecular force and extended the structure of the protein.The results of CD chromatography showed that both enzymatic hydrolysis and multi-frequency ultrasonic treatment changed the secondary structure of the protein,mainly represented by the decrease of?-helix content and the increase of?-sheet content.SEM results showed that the enzymatic hydrolysis and multi-frequency ultrasonic treatment both changed the apparent structure of the protein,the surface was rough,and the voids was increased.AFM results showed that the enzymatic hydrolysis reaction loosened the microstructure of the protein.Multi-frequency ultrasonic treatment caused the aggregation of protein particles and the particle diameter became larger.The above-mentioned changes of protein structure will be beneficial to its contact with the protease and improve the efficiency of enzymatic hydrolysis.The NIR spectroscopy of protease indicated that the enzymatic hydrolysis made the structure change of protease and became extended.Multi-frequency ultrasonic treatment made the protease structure more active.SEM results showed that the enzymatic hydrolysis and multi-frequency ultrasonic treatment both made the apparent structure of the protease rough and the specific surface area increased.The above changes of protease structure will facilitate its contact with the protein and the increase of enzymatic hydrolysis efficiency.The amino acid composition of the product corn peptide showed that the hydrophobic amino acid content was increased by 4.6%after multi-frequency ultrasonic treatment.The results of the changes of the proteins,immobilized enzymes and product peptides under the ultrasonic field can effectively explain the reason of the increases in the peptide concentration and ACE inhibitory rate under the preparation method with coupled immobilized enzymolysis and membrane separation assisted by multi-frequency ultrasound.(3)During the screening of six spectral pretreatment methods,it was found that Baseline method was more suitable for pretreatment of the peptide concentration spectrum.The SNV was more suitable for pretreatment of the ACE inhibition rate spectrum.The established CARS-PLS prediction model of peptide concentration had the best performance,with R_c=0.972,RMSEC=0.07,R_p=0.970,RMSEP=0.07.The established UVE-PLS prediction model of ACE inhibition rate had the best performance,with R_c=0.935,RMSEC=0.32,R_p=0.911,RMSEP=0.46.Both models can greatly reduce the number of redundant spectra and improve the prediction performance.